In vivo reorganization of the actin cytoskeleton in leaves of Nicotiana tabacum L. transformed with plastin-GFP. Correlation with light-activated chloroplast responses

<p>Abstract</p> <p>Background</p> <p>The actin cytoskeleton is involved in the responses of plants to environmental signals. Actin bundles play the role of tracks in chloroplast movements activated by light. Chloroplasts redistribute in response to blue light in the mesophyll cells of <it>Nicotiana tabacum</it>. The aim of this work was to study the relationship between chloroplast responses and the organization of actin cytoskeleton in living tobacco cells. Chloroplast movements were measured photometrically as changes in light transmission through the leaves. The actin cytoskeleton, labeled with plastin-GFP, was visualised by confocal microscopy.</p> <p>Results</p> <p>The actin cytoskeleton was affected by strong blue and red light. No blue light specific actin reorganization was detected. EGTA and trifluoperazine strongly inhibited chloroplast responses and disrupted the integrity of the cytoskeleton. This disruption was reversible by Ca²⁺or Mg²⁺. Additionally, the effect of trifluoperazine was reversible by light. Wortmannin, an inhibitor of phosphoinositide kinases, potently inhibited chloroplast responses but did not influence the actin cytoskeleton at the same concentration. Also this inhibition was reversed by Ca²⁺and Mg²⁺. Magnesium ions were equally or more effective than Ca²⁺in restoring chloroplast motility after treatment with EGTA, trifluoperazine or wortmannin.</p> <p>Conclusion</p> <p>The architecture of the actin cytoskeleton in the mesophyll of tobacco is significantly modulated by strong light. This modulation does not affect the direction of chloroplast redistribution in the cell. Calcium ions have multiple functions in the mechanism of the movements. Our results suggest also that Mg²⁺is a regulatory molecule cooperating with Ca²⁺in the signaling pathway of blue light-induced tobacco chloroplast movements.</p

SnRK2.10 kinase differentially modulates expression of hub WRKY transcription factors genes under salinity and oxidative stress in Arabidopsis thaliana

In nature, all living organisms must continuously sense their surroundings and react to the occurring changes. In the cell, the information about these changes is transmitted to all cellular compartments, including the nucleus, by multiple phosphorylation cascades. Sucrose Non-Fermenting 1 Related Protein Kinases (SnRK2s) are plant-specific enzymes widely distributed across the plant kingdom and key players controlling abscisic acid (ABA)-dependent and ABA independent signaling pathways in the plant response to osmotic stress and salinity. The main deleterious effects of salinity comprise water deficiency stress, disturbances in ion balance, and the accompanying appearance of oxidative stress. The reactive oxygen species (ROS) generated at the early stages of salt stress are involved in triggering intracellular signaling required for the fast stress response and modulation of gene expression. Here we established in Arabidopsis thaliana that salt stress or induction of ROS accumulation by treatment of plants with H2 O 2 or methyl viologen (MV) induces the expression of several genes encoding transcription factors (TFs) from the WRKY DNA-Binding Protein (WRKY) family. Their induction by salinity was dependent on SnRK2.10, an ABA non-activated kinase, as it was strongly reduced in snrk2.10 mutants. The effect of ROS was clearly dependent on their source. Following the H2 O 2 treatment, SnRK2.10 was activated in wild-type (wt) plants and the induction of the WRKY TFs expression was only moderate and was enhanced in snrk2.10 lines. In contrast, MV did not activate SnRK2.10 and the WRKY induction was very strong and was similar in wt and snrk2.10 plants. A bioinformatic analysis indicated that the WRKY33, WRKY40, WRKY46, and WRKY75 transcription factors have a similar target range comprising numerous stress-responsive protein kinases. Our resultsindicate that the stress-related functioning of SnRK2.10 is fine-tuned by the source and intracellular distribution of ROS and the co-occurrence of other stress factors

SNF1-related protein kinases type 2 are involved in plant responses to cadmium stress

Cadmium ions are notorious environmental pollutants. In order to adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SNF1-related protein kinases 2 (SnRK2s) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of Nicotiana tabacum Osmotic Stress-Activated Protein Kinase (NtOSAK) activity in tobacco BY-2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an L-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homologue of NtOSAK in Arabidopsis thaliana. Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins in roots of plants exposed to Cd2+ showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the phytochelatins level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd2+ treatment. Our data show significantly lower Cd2+-induced ROS accumulation in the mutants’ roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions

Arabidopsis thaliana Nudix hydrolase AtNUDT7 forms complexes with the regulatory RACK1A protein and Ggamma subunits of the signal transducing heterotrimeric G protein.

Arabidopsis thaliana AtNUDT7 Nudix pyrophosphatase hydrolyzes NADH and ADP-ribose in vitro and is an important factor in the cellular response to diverse biotic and abiotic stresses. Several studies have shown that loss-of-function Atnudt7 mutant plants display many profound phenotypes. However the molecular mechanism of AtNUDT7 function remains elusive. To gain a better understanding of this hydrolase cellular role, proteins interacting with AtNUDT7 were identified. Using AtNUDT7 as a bait in an in vitro binding assay of proteins derived from cultured Arabidopsis cell extracts we identified the regulatory protein RACK1A as an AtNUDT7-interactor. RACK1A-AtNUDT7 interaction was confirmed in a yeast two-hybrid assay and in a pull-down assay and in Bimolecular Fluorescence Complementation (BiFC) analysis of the proteins transiently expressed in Arabidopsis protoplasts. However, no influence of RACK1A on AtNUDT7 hydrolase catalytic activity was observed. In vitro interaction between RACK1A and the AGG1 and AGG2 gamma subunits of the signal transducing heterotrimeric G protein was also detected and confirmed in BiFC assays. Moreover, association between AtNUDT7 and both AGG1 and AGG2 subunits was observed in Arabidopsis protoplasts, although binding of these proteins could not be detected in vitro. Based on the observed interactions we conclude that the AtNUDT7 Nudix hydrolase forms complexes in vitro and in vivo with regulatory proteins involved in signal transduction. Moreover, we provide the initial evidence that both signal transducing gamma subunits bind the regulatory RACK1A protein

The SnRK2.10 kinase mitigates the adverse effects of salinity by protecting photosynthetic machinery

SNF1-Related protein kinases Type 2 (SnRK2) are plant-specific enzymes widely distributed across the plant kingdom. They are key players controlling abscisic acid (ABA)-dependent and ABA-independent signaling pathways in the plant response to osmotic stress. Here we established that SnRK2.4 and SnRK2.10, ABA-nonactivated kinases, are activated in Arabidopsis thaliana rosettes during the early response to salt stress and contribute to leaf growth retardation under prolonged salinity but act by maintaining different salt-triggered mechanisms. Under salinity, snrk2.10 insertion mutants were impaired in the reconstruction and rearrangement of damaged core and antenna protein complexes in photosystem II (PSII), which led to stronger non-photochemical quenching, lower maximal quantum yield of PSII, and lower adaptation of the photosynthetic apparatus to high light intensity. The observed effects were likely caused by disturbed accumulation and phosphorylation status of the main PSII core and antenna proteins. Finally, we found a higher accumulation of reactive oxygen species (ROS) in the snrk2.10 mutant leaves under a few-day-long exposure to salinity which also could contribute to the stronger damage of the photosynthetic apparatus and cause other deleterious effects affecting plant growth. We found that the snrk2.4 mutant plants did not display substantial changes in photosynthesis. Overall, our results indicate that SnRK2.10 is activated in leaves shortly after plant exposure to salinity and contributes to salt stress tolerance by maintaining efficient photosynthesis and preventing oxidative damage. Radosław Mazur, Justyna Maszkowska, Anna Anielska-Mazur, Maciej Garstka, Lidia Polkowska-Kowalczyk, Anna Czajkowska, Agnieszka Zmienko, Grazyna Dobrowolska, Anna Kuli

Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity

International audienceSeveral studies focused on elucidating the mechanism of nitric oxide (NO) signalling in plant cells highlighted that its biological effects are partly mediated by protein kinases. Identity of these kinases and details of how NO modulates their activities, however, remain poorly investigated. Here, we have attempted to clarify the mechanisms underlying NO action in regulation of Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SNF1-related protein kinase 2 family. We found that in tobacco BY-2 cells exposed to salt stress, NtOSAK is rapidly activated partly through a NO-dependent process. This activation, as well as the one observed following treatment of BY-2 cells with the NO donor DEA/NO, involved the phosphorylation of two residues located in the kinase activation loop, one being identified as Ser-158. Our results indicate that NtOSAK does not undergo the direct chemical modifications of its Cys residues by S-nitrosylation. Using a co-immunoprecipitation-based strategy, we identified several proteins present in immunocomplex with NtOSAK in salt-treated cells including the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Our results indicate that NtOSAK directly interacts with GAPDH in planta. Furthermore, in response to salt, GAPDH showed a transient increase of its S-nitrosylation level which was correlated with the time course of NtOSAK activation. However, GADPH S-nitrosylation did not influence its interaction with NtOSAK and did not impact the activity of the protein kinase. Taken together, the results support the hypothesis that NtOSAK and GAPDH form a cellular complex and that both proteins are regulated directly or indirectly by NO

SNF1-related Protein Kinases 2 Are Negatively Regulated by a Plant-specific Calcium Sensor*

SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb3+ as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 105 m−1. The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity